1. Field of the Invention
The present invention relates to a .beta.-catotene hydroxylase, a DNA coding for the .beta.-catotene hydroxylase, a recombinant vector comprising the DNA, a transformant transformed with the vector, a method for preparing the .beta.-catotene hydroxylase and a method for preparing .beta.-cryptoxanthin.
2. Description of the Prior Art
In carotenoids synthesized by animals, plants and microorganisms, there are a group of compounds with a hydroxyl group(s) generically called xanthophyll. These compounds are generated from carotenoids (starting substances) by the catalytic action of hydroxylase. For example, one hydroxyl group is introduced into .beta.-carotene to yield .beta.-cryptoxanthin, into which another hydroxyl group is introduced to yield zeaxanthin via the biosynthetic pathway shown below (see arrow (1) in FIG. 1):
.beta.-carotene .fwdarw..beta.-cryptoxanthin .fwdarw.zeaxanthin PA1 (a) a protein consisting of the amino acid sequence as shown in SEQ ID NO: 2; PA1 (b) a protein which consists of the amino acid sequence as shown in SEQ ID NO: 2 having deletion, substitution or addition of one or several amino acids and which has .beta.-carotene hydroxylase activity. PA1 (a) a protein consisting of the amino acid sequence as shown in SEQ ID NO: 2; PA1 (b) a protein which consists of the amino acid sequence as shown in SEQ ID NO: 2 having deletion, substitution or addition of one or several amino acids and which has .beta.-carotene hydroxylase activity.
This .beta.-cryptoxanthin is obtained by introducing a hydroxyl group into one of the two ionone rings present in .beta.-carotene. When another hydroxyl group is introduced into a position symmetric to the former position, zeaxanthin is produced (FIG. 1).
In a large number of plants and microorganisms, metabolism proceeds from .beta.-carotene to zeaxanthin, producing little .beta.-cryptoxanthin, the intermediate into which only one hydroxyl group is introduced.
This reaction is controlled by a hydroxylase gene called Crt Z. In this enzyme reaction, it is considered that two hydroxyl groups are introduced almost simultaneously. For example, under the control of a hydroxylase gene cloned from a bacterium belonging to the genus Erwinia, zeaxanthin is produced which is obtainable by introducing two hydroxyl groups into .beta.-carotene.
In Citrus unshiu (Satsuma mandarine) which is a major citrus fruit in Japan, .beta.-cryptoxanthin obtainable by introducing one hydroxyl group into .beta.-carotene is considered to be one of the most important carotenoids. In particular, .beta.-cryptoxanthin occupies 60-70% of the total carotenoid content in the edible part of this fruit.
Considering this high .beta.-cryptoxanthin content of Citrus unshiu, it is hard to think that the .beta.-cryptoxanthin in Citrus unshiu is produced by a gene involved in the above-mentioned metabolic pathway. Also, it is still unknown whether .beta.-cryptoxanthin is produced by those genes which have been already cloned.